Chimeric promoter molecules for gene expression in prokaryotes

ABSTRACT

The present invention provides regulatory polynucleotide molecules isolated from a 16S rDNA for enhanced expression of heterologous genes. The invention further discloses compositions, polynucleotide constructs, transformed host cells containing the regulatory polynucleotide sequences, and methods for preparing and using the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. Provisional ApplicationSer. No. 61/035,255 filed Mar. 10, 2008, the entire disclosure of whichis incorporated herein by reference.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING IN COMPUTER READABLE FORM

The Sequence Listing, which is a part of the present disclosure,includes a computer readable form 18.9 KB file entitled“MONS196US_ST25.txt” comprising nucleotide sequences of the presentinvention. The subject matter of the Sequence Listing is incorporatedherein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of molecular biology andgenetic engineering, and polynucleic acid molecules useful for geneexpression in prokaryotes. Specifically, the present invention discloseschimeric polynucleic acid molecules comprising promoter activity inbacterial cells. The invention further discloses DNA constructs andbacterial cells comprising the polynucleic acid molecules, and methodsof producing and using the same.

2. Description of Related Art

One of the goals of genetic engineering is to produce organisms withdesirable characteristics or traits. The proper expression of adesirable transgene in a transgenic organism is one way to achieve thisgoal. Elements having gene regulatory activity, i.e. regulatory elementssuch as promoters, leaders, and transcription termination regions, arenon-coding polynucleotide molecules that play an integral part in theoverall expression of genes in living cells. Regulatory elements thatfunction in prokaryotes are therefore useful for modifying theirphenotypes through the methods of genetic engineering.

While previous work has provided a number of regulatory elements usefulto affect gene expression in transgenic bacteria, there is still a greatneed for novel regulatory elements with beneficial expressioncharacteristics. Many previously identified regulatory elements fail toprovide the patterns or levels of expression required to fully realizethe benefits of expression of selected genes in transgenic bacteria. Oneexample of this is the need for regulatory elements capable of drivingstrong gene expression in different types of bacteria.

A promoter is a key element for directing gene expression in a cell. Thetranscription machinery is assembled and transcription is initiated fromthe promoter DNA molecule. Transcription factors influence the strengthof a transcript from a promoter molecule. Accordingly, regions withinthe promoter molecule function to enhance or repress transcription.

The genetic enhancement of bacteria provides significant benefits tosociety. For example, bacteria may be enhanced with a transgene toprovide desirable biosynthetic, commercial, chemical, insecticidal,industrial, nutritional, or pharmaceutical properties. Despite theavailability of many molecular tools, however, the genetic modificationof bacteria is often constrained by an insufficient expression of theengineered transgene.

High level gene expression requires a strong 5′ regulatory sequence(promoter) and may also be affected by sequences found 3′ to a codingsequence. Currently only a limited number of strong promoters areavailable from prokaryotes. Thus, there is a need for additionalpromoters that are useful for expressing genes, especially single copygenes in a single or low copy vector.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a polynucleotide moleculecomprising a 16S rDNA promoter molecule operably linked to a nucleicacid comprising a heterologous ribosomal binding site, wherein thepolynucleotide molecule has promoter activity. The 16S rDNA promotermolecule may be isolated from a prokaryote. In specific embodiments, the16S rDNA promoter molecule comprises a nucleic acid sequence selectedfrom the group consisting of: a) a nucleic acid sequence comprising SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, or any of SEQ IDNOs:22-37; b) a nucleic acid sequence comprising at least 65% sequenceidentity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, orany of SEQ ID NOs:22-37, wherein the nucleic acid sequence comprisespromoter activity; and c) a fragment of the nucleic acid sequence of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, or any of SEQ IDNOs:22-37, wherein the fragment has promoter activity. In furtherembodiments, the prokaryote is a bacterium, and may be a member of theRhizobiales further including, for example, Rhizobium spp.,Sinorhizobium spp., Mesorhizobium spp., Phyllobacterium spp.,Ochrobactrum spp., and Bradyrhizobium spp. In still further embodiments,the ribosomal binding site may be isolated from the Agrobacterium virEoperon.

In another aspect, a polynucleotide molecule of the invention may bedefined as comprising a sequence selected from the group consisting of:a) a nucleic acid sequence comprising SEQ ID NO:6, SEQ ID NO:7 or SEQ IDNO:8; b) a nucleic acid sequence comprising at least 65% sequenceidentity to SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8; and c) a fragmentof the nucleic acid sequence of SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.In particular embodiments, the polynucleotide molecule may comprise atleast about 65%, at least about 85%, at least about 90%, at least about95% identity, or at least about 98% identity to the nucleic acidsequence of SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8. The polynucleotidemolecule may comprise the sequence of SEQ ID NO:6, SEQ ID NO:7 or SEQ IDNO:8. The polynucleotide molecule may also be defined as comprising afragment of the nucleic acid sequence of SEQ ID NO:6, SEQ ID NO:7 or SEQID NO:8. In further embodiments, the polynucleotide molecule may beoperably linked to a heterologous transcribable polynucleotide molecule,including, for example, a heterologous transcribable polynucleotidemolecule that encodes a selectable marker. Examples of such selectablemarkers include those that confer resistance to a selective agentselected from the group consisting of: kanamycin, spectinomycin,streptomycin, hygromycin, gentamycin, glyphosate, dicamba, andglufosinate. In one embodiment, the selectable marker is aadA.

In yet another aspect, the invention provides a transgenic celltransformed with a polynucleotide described herein. In specificembodiments, the cell is a prokaryotic cell, and may be a bacterialcell, including a member of the Rhizobiales. The Rhizobiales may beselected from the group consisting of: Rhizobium spp., Sinorhizobiumspp., Mesorhizobium spp., Phyllobacterium spp., Ochrobactrum spp., andBradyrhizobium spp. In one embodiment, the bacterial cell is an E. colicell.

In still yet another aspect, the invention provides a transgenicorganelle comprising the polynucleotide molecule of claim 1.

Still further provided by the invention is a recombinant Agrobacteriumcell wherein the function of the native virE operon promoter of the cellhas been replaced with a heterologous constitutive promoter. The cellmay be an Agrobacterium tumefaciens cell. In specific embodiments, theheterologous promoter may comprise a promoter sequence provided herein.

Another aspect of the invention provides a method for enhancingexpression of a transgene in a prokaryotic cell comprising: (a)transforming the cell with a transgene operably linked to a 16S rDNApromoter operably linked to a ribosomal binding site; (b) growing thecell; and (c) testing the cell for enhanced expression of the transgene.In one embodiment, the transgene confers a commercially important trait.In another embodiment, the method further comprises the step of: (d)harvesting a product of the enhanced expression of the transgene. In yetanother embodiment of the method, the product of the enhanced transgeneexpression is a protein or a compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Alignment of Agrobacterium tumefaciens C58 16S rDNA promotersequences. Arrow under G indicates the isolated promoter's starting basepair. Arrow under A indicates potential 16S rRNA initiation site; aHindIII site is underlined.

FIG. 2. The long P-rrn promoter.

FIG. 3. The short P-rrn promoter.

FIG. 4. The long P-rrn promoter with filled-in HindIII site(underlined).

FIG. 5. Diagrammatic representation of plasmid pMON96945.

FIG. 6. Diagrammatic representation of plasmid pMON107330. pMON107330 isa 1 T-DNA low copy oriRi vector carrying the long P-rrn with aadA whichconfers on Agrobacterium the ability to tolerate spectinomycinconcentration over 300 mg/l, causing the culture to grow faster than aculture containing cells with a multiple copy oriV vector.

FIG. 7. Diagrammatic representation of plasmid pMON107331. pMON107331 isa 1 T-DNA low copy oriRi vector carrying the short P-rrn with aadA whichconfers the same spectinomycin resistance as the long P-rrn with aadA inE. coli but in Agrobacterium the resistance substantially is reduced.

FIG. 8. Diagrammatic representation of plasmid pMON107336. pMON107336 isa 2 T-DNA low copy oriRi vector carrying the long P-rrn with aadA.

FIG. 9. Diagrammatic representation of plasmid pMON107337. pMON107337 isa 2 T-DNA low copy oriRi vector carrying the HindIII minus P-rrn withaadA.

FIG. 10. Growth rates of E. coli TOP10 cells carrying vectors comprisingP-rrn-aadA expression cassette. pMON101343 with oriV replication originfor Agrobacterium replication and aadA with native aadA promoter,pMON107330 with oriRi replication origin for Agrobacterium replicationwith long P-rrn-aadA (P-rrn L), and pMON107331 with oriRi replicationorigin for Agrobacterium replication with short P-rrn-aadA (P-rrn S).

FIG. 11. Growth rates of Agrobacterium tumefaciens ABI cells carryingvectors comprising P-rrn-aadA expression cassettes. pMON101343 with oriVreplication origin for Agrobacterium replication and aadA with itsnative aadA promoter, pMON107330 with oriRi replication origin forAgrobacterium replication with long P-rrn-aadA (P-rrn L), and pMON107331with oriRi replication origin for Agrobacterium replication with shortP-rrn-aadA (P-rrn S).

FIG. 12. Growth rates of Agrobacterium tumefaciens ABI cells carryingvectors comprising P-rrn-aadA expression cassettes. pMON101343 with oriVreplication origin for Agrobacterium replication and aadA with itsnative aadA promoter, pMON107330 with oriRi replication origin forAgrobacterium replication with long P-rrn-aadA (P-rrn L), and pMON107337with oriRi replication origin for Agrobacterium replication with longP-rrn-aadA (P-rrn L) with filled-in HindIII site.

DESCRIPTION OF SEQUENCE LISTING

A summary of sequences referred to herein is provided below.

Identified from SEQ GenBank ID NO Organism/source Details From AccessionNo. 1 Agrobacterium tumefaciens Identified enhancer and gDNA AE009201,C58 promoter element AE008688 2 Agrobacterium tumefaciens Identifiedenhancer and gDNA AE008980, C58 promoter element AE008688 3Agrobacterium tumefaciens Identified enhancer and gDNA AE009324, C58promoter element AE008689 4 Agrobacterium tumefaciens Identifiedenhancer and gDNA AE009348, C58 promoter element AE008689 5Agrobacterium tumefaciens Consensus of SEQ ID N/A N/A C58 NOs: 1-4 6Synthetic Synthetic promoter N/A N/A (En + Pro + RBS + ATG) sequence 7Synthetic (Pro + RBS + ATG) Synthetic promoter N/A N/A sequence 8Synthetic Synthetic promoter N/A N/A (En + Pro + RBS + ATG − sequenceHindIII site)  9-21 Primers for amplification, Synthetic N/A N/Acloning, and/or analysis of P-rrn sequences and constructs 22-25Rhizobium leguminosarum Identified enhancer and gDNA NC_008380 bv.viciae 3841 promoter element and consensus sequence 26-29 Rhizobium etliCFN 42 Identified enhancer and gDNA NC_007761 promoter element andconsensus sequence 30-33 Sinorhizobium medicae Identified enhancer andgDNA CP000738 WSM419 promoter element and consensus sequence 34-37Sinorhizobium meliloti Identified enhancer and gDNA NC_003047 1021promoter element and consensus sequence

DETAILED DESCRIPTION OF THE INVENTION

A strong prokaryotic promoter (P-rrn) from the 16S rDNA gene of A.tumefaciens C58 strain which specifies a high level of gene expressionin both E. coli and Agrobacterium has now been identified andcharacterized. The isolated promoter is useful for over-expressinggenes, for example, in E. coli and Agrobacterium, conferring a highlevel of gene expression in low copy vectors, reducing cloning time andquality control time due to faster growth, and reducing Agrobacteriumgrowth variations due to low copy vectors.

The P-rrn promoter from Agrobacterium C58 when fused to the aadA geneconfers in both E. coli and Agrobacterium high level resistance tospectinomycin. In E. coli, P-rrn-aadA vectors allow harvesting of cellsin 5-6 hours, while use of a native aadA promoter requires about 8hours. The enhanced growth of E. coli containing a vector comprising theP-rrn promoter can speed up the cloning process. In Agrobacterium, aP-rrn-aadA construct confers resistance to application of spectinomycinat over 300 mg/L. In the range of spectinomycin level of 75-300 mg/l,Agrobacterium cultures with the oriRi vectors with P-rrn-aadA showed0.1-0.2 higher ODs at OD600 after overnight culture compared tomulticopy oriV vectors.

The invention thus provides polynucleotide molecules having generegulatory activity. In one embodiment, examples of such sequences areprovided by the chimeric polynucleotides of SEQ ID NOs: 6-8. Suchpolynucleotide molecules are capable of directing the expression of anoperably linked transcribable polynucleotide molecule in bacterialcells. The present invention also provides methods of modifying,producing, and using the same. The invention further providescompositions, transformed host cells, and methods for preparing andusing the same.

The invention describes the identification and isolation of a 16S rRNApromoter (P-rrn) from a member of the Rhizobiales, and more specificallyAgrobacterium tumefaciens and describes the features of this promoter.The promoter has been shown to exhibit a variety of properties. One ofthe properties described herein is the identification of a segment ofthe P-rrn promoter that enhances growth of E. coli and Agrobacteriumtumefaciens cells under selection pressure.

The invention also provides, in specific embodiments, organellestransformed with a polynucleotide sequence described herein. Methods forthe creation of transgenic organelles, including plant plastids, arewell known in the art and described in, for example U.S. Pat. Nos.6,492,578 and 6,218,145.

Polynucleotide Molecules

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

The phrases “coding sequence,” “structural sequence,” and “transcribablepolynucleotide sequence” refer to a physical structure comprising anorderly and contiguous linear arrangement of nucleic acids. The nucleicacids are arranged in a series of nucleic acid triplets that each form acodon when viewed within a particular reading frame along the length ofthe contiguous linear arrangement of nucleic acids. Each codon encodesfor a specific amino acid. Thus the coding sequence, structuralsequence, and transcribable polynucleotide sequence encode a series ofamino acids forming a protein, polypeptide, or peptide sequence. The“coding sequence,” “structural sequence,” and “transcribablepolynucleotide sequence” encode a series of amino acids forming aprotein, polypeptide, or peptide sequence. The coding sequence,structural sequence, and transcribable polynucleotide sequence may becontained, without limitation, within a larger nucleic acid molecule,vector, etc. In addition, the orderly arrangement of nucleic acids inthese sequences may be depicted, without limitation, in the form of asequence listing, figure, table, electronic medium, and the like.

As used herein, the term “polynucleotide molecule or polynucleic acidmolecule” refers to the single- or double-stranded DNA or RNA moleculeof genomic or synthetic origin, i.e., a polymer of deoxyribonucleotideor ribonucleotide bases, respectively, read from the 5′ (upstream) endto the 3′ (downstream) end.

As used herein, the term “polynucleotide sequence” refers to thesequence of a polynucleotide molecule. The nomenclature for nucleotidebases as used herein can be found in Part 1 of Title 37 of the UnitedStates Code of Federal Regulations, in particular at section 1.822 isused herein.

As used herein, the term “regulatory element” refers to a polynucleotidemolecule having gene regulatory activity, i.e. one that has the abilityto affect the transcription or translation of an operably linkedtranscribable polynucleotide molecule. Regulatory elements such aspromoters, leaders, introns, and transcription termination regions arepolynucleotide molecules having gene regulatory activity which play anintegral part in the overall expression of genes in living cells.Isolated regulatory elements that function in bacteria are thereforeuseful for modifying their phenotypes through the methods of geneticengineering. By “regulatory element” is intended a series of nucleotidesthat determines if, when, and at what level a particular gene isexpressed. The regulatory DNA sequences specifically interact withregulatory proteins or other proteins.

By “promoter activity” it is meant that a nucleic acid is capable ofserving as a promoter element and thus directing the transcription of anoperably linked transcribable polynucleotide molecule.

As used herein, the term “operably linked or linked” refers to a firstpolynucleotide molecule joined to a second polynucleotide wherein thepolynucleotide molecules are so arranged that the first polynucleotidemolecule affects the function of the second polynucleotide molecule. Thetwo polynucleotide molecules may be part of a single contiguouspolynucleotide molecule and may be adjacent. In some embodiments apromoter enhancer is operably linked to a promoter and/or a promoter isoperably linked to a polynucleotide of interest so that the promotermodulates transcription of the linked polynucleotide molecule ofinterest in a cell.

As used herein, the term “gene regulatory activity” refers to apolynucleotide molecule capable of affecting transcription ortranslation of an operably linked polynucleotide molecule. An isolatedpolynucleotide molecule having gene regulatory activity may providetemporal expression or modulate levels and rates of expression of theoperably linked transcribable polynucleotide molecule. An isolatedprokaryotic polynucleotide molecule having gene regulatory activity maycomprise a promoter, ribosomal binding site, additional transcriptionfactor binding site (s), or 3′ transcriptional termination region.

As used herein, the term “gene expression” or “expression” refers to thetranscription of a DNA molecule into a transcribed RNA molecule. Geneexpression may be described as related to temporal, quantitative orqualitative indications. The transcribed RNA molecule may be translatedto produce a protein molecule or may provide an antisense or otherregulatory RNA molecule, such as a dsRNA, a tRNA, a rRNA, and the like.

As used herein, an “expression pattern” is any pattern of differentialgene expression. In certain embodiments, an expression pattern may becharacterized as one or more of temporal, stress, environmental,physiological, pathological, cell cycle, and/or chemically responsiveexpression patterns.

As used herein, the term “transcribable polynucleotide molecule” refersto any polynucleotide molecule capable of being transcribed into a RNAmolecule, including but not limited to protein coding sequences andsequences useful for gene suppression.

A “transgene” comprises a DNA molecule heterologous to a host cell.

Determination of Sequence Similarity Using Hybridization Techniques

Nucleic acid hybridization is a technique well known to those of skillin the art of DNA manipulation. The hybridization properties of a givenpair of nucleic acids are an indication of their similarity or identity.

The term “hybridization” refers generally to the ability of nucleic acidmolecules to join via complementary base strand pairing. Suchhybridization may occur when nucleic acid molecules are contacted underappropriate conditions. “Specifically hybridizes” refers to the abilityof two nucleic acid molecules to form an anti-parallel, double-strandednucleic acid structure. A nucleic acid molecule is said to be the“complement” of another nucleic acid molecule if they exhibit “completecomplementarity,” i.e., each nucleotide in one molecule is complementaryto its base pairing partner nucleotide in another molecule. Twomolecules are said to be “minimally complementary” if they can hybridizeto one another with sufficient stability to permit them to remainannealed to one another under at least conventional “low-stringency”conditions. Similarly, the molecules are said to be “complementary” ifthey can hybridize to one another with sufficient stability to permitthem to remain annealed to one another under conventional“high-stringency” conditions. Nucleic acid molecules that hybridize toother nucleic acid molecules, e.g., at least under low stringencyconditions are said to be “hybridizable cognates” of the other nucleicacid molecules. Conventional low stringency and high stringencyconditions are described herein and by Sambrook et al., (MolecularCloning: A Laboratory Manual 2^(nd) Ed., Cold Spring Harbor LaboratoryPress, 1989) herein referred to as Sambrook et al., 1989, and by Haymeset al., (Nucleic Acid Hybridization, A Practical Approach, IRL Press,Washington, D.C., 1985). Departures from complete complementarity arepermissible, as long as such departures do not completely preclude thecapacity of the molecules to form a double-stranded structure.

Low stringency conditions may be used to select nucleic acid sequenceswith lower sequence identities to a target nucleic acid sequence. Onemay wish to employ conditions such as about 0.15 M to about 0.9 M sodiumchloride, at temperatures ranging from about 20° C. to about 55° C. Highstringency conditions may be used to select for nucleic acid sequenceswith higher degrees of identity to the disclosed nucleic acid sequences(Sambrook et al., 1989). High stringency conditions typically involvenucleic acid hybridization in about 2× to about 10×SSC (diluted from a20×SSC stock solution containing 3 M sodium chloride and 0.3 M sodiumcitrate, pH 7.0 in distilled water), about 2.5× to about 5×Denhardt'ssolution (diluted from a 50× stock solution containing 1% (w/v) bovineserum albumin, 1% (w/v) Ficoll, and 1% (w/v) polyvinylpyrrolidone indistilled water), about 10 mg/mL to about 100 mg/mL fish sperm DNA, andabout 0.02% (w/v) to about 0.1% (w/v) SDS, with an incubation at about50° C. to about 70° C. for several hours to overnight. High stringencyconditions are preferably provided by 6×SSC, 5×Denhardt's solution, 100mg/mL fish sperm DNA, and 0.1% (w/v) SDS, with an incubation at 55° C.for several hours. Hybridization is generally followed by several washsteps. The wash compositions generally comprise 0.5× to about 10×SSC,and 0.01% (w/v) to about 0.5% (w/v) SDS with a 15 minute incubation atabout 20° C. to about 70° C. Preferably, the nucleic acid segmentsremain hybridized after washing at least one time in 0.1×SSC at 65° C.

A chimeric promoter polynucleic acid molecule of the present inventionpreferably comprises a P-rrn promoter polynucleic acid sequence thathybridizes, under low or high stringency conditions, with any of SEQ IDNOs: 6-8, any complements thereof, or any fragments thereof, or any ciselements thereof. In a particular embodiment, the invention provides asegment of the P-rrn promoter that allows for growth of E. coli orAgrobacterium cells in the presence of a selective agent, wherein apolynucleotide sequence encoding a gene product that specifies toleranceor resistance to the selective agent is operably linked to the P-rrnpromoter.

In another embodiment, the invention provides a segment of a P-rrnpromoter that drives the expression of a transgene, for instance in aprokaryote such as E. coli, that results in production of a protein ofcommercial importance or whose product results in production of acommercially important compound. In other embodiments, the promoters ofthe present invention are used to replace native promoters of certaingenes such as those of one or more of the virB operon, virC operon, virDoperon, and/or virE operon in a host organism such as Agrobacterium toenhance expression of these genes. Enhanced expression of the virBoperon, virC operon, and virD operon is known to result in enhanced celltransformation frequency by Agrobacterium, and enhanced expression ofthe virE operon is known to provide for more efficient transfer of largeT-DNA segments to cells.

Analysis of Sequence Similarity Using Identity Scoring

As used herein, the term “sequence identity” refers to the extent towhich two optimally aligned polynucleotide sequences are identical. Anoptimal sequence alignment is created by manually aligning twosequences, e.g. a reference sequence and another sequence, to maximizethe number of nucleotide matches in the sequence alignment withappropriate internal nucleotide insertions, deletions, or gaps.

As used herein, the term “percent sequence identity” or “percentidentity” or “% identity” is the identity fraction times 100. The“identity fraction” for a sequence optimally aligned with a referencesequence is the number of nucleotide matches in the optimal alignment,divided by the total number of nucleotides in the reference sequence,e.g. the total number of nucleotides in the full length of the entirereference sequence. Thus, one embodiment of the invention is a DNAmolecule comprising a sequence that when optimally aligned to areference sequence, for instance provided herein as any of SEQ IDNOs:1-8 and SEQ ID NOs:22-37, has at least 65 percent identity orhigher, has at least 70 percent identity or higher, has at least 75percent identity or higher, has at least 80 percent identity or higher,about 85 percent identity or higher, about 90 percent identity orhigher, about 95 percent identity or higher, or at least 96 percentidentity, 97 percent identity, 98 percent identity, or 99 percentidentity to the reference sequence, and has gene regulatory activity.

As used herein, the term “substantial percent sequence identity” refersto a percent sequence identity of at least about 70% sequence identity,at least about 80% sequence identity, at least about 85% identity, atleast about 90% sequence identity, or even greater sequence identity,such as about 98% or about 99% sequence identity. Thus, one embodimentof the invention is a polynucleotide molecule that has at least about70% sequence identity, at least about 80% sequence identity, at leastabout 85% identity, at least about 90% sequence identity, or evengreater sequence identity, such as at least about 95%, 98% or 99%sequence identity with a polynucleotide sequence described herein.Polynucleotide molecules and variants thereof that are capable ofregulating transcription of operably linked transcribable polynucleotidemolecules and have a substantial percent sequence identity to thepolynucleotide sequences of the polynucleotide molecules provided hereinare encompassed within the scope of this invention.

“Homology” refers to the level of similarity between two or more nucleicacid or amino acid sequences in terms of percent of positional identity(i.e., sequence similarity or identity). Homology also refers to theconcept of similar functional properties among different nucleic acidsor proteins

Polynucleotide Molecules, Motifs, Fragments, Chimeric Molecules,Enhancers

As used herein, the term “enhancer domain” refers to a cis-actingtranscriptional regulatory element (cis-element), which confers anaspect of the overall modulation of gene expression. An enhancer domainmay function to bind transcription factors, trans-acting protein factorsthat regulate transcription. Some enhancer domains bind more than onetranscription factor, and transcription factors may interact withdifferent affinities with more than one enhancer domain. Enhancerdomains can be identified by a number of techniques, including deletionanalysis, i.e., deleting one or more nucleotides from the 5′ end orinternal to a promoter; DNA binding protein analysis using DNase Ifootprinting, methylation interference, electrophoresis mobility-shiftassays, in vivo genomic footprinting by ligation-mediated PCR, and otherconventional assays; or by DNA sequence similarity analysis with knowncis-element motifs by conventional DNA sequence comparison methods. Thefine structure of an enhancer domain can be further studied bymutagenesis (or substitution) of one or more nucleotides or by otherconventional methods. Enhancer domains can be obtained by chemicalsynthesis or by isolation from regulatory elements that include suchelements, and they can be synthesized with additional flankingnucleotides that contain useful restriction enzyme sites to facilitatesubsequence manipulation. Any of the polynucleotide molecules of thepresent invention may comprise an enhancer.

Chimeric promoter molecules of the present invention combine a 16S rDNAenhancer molecule, a P-rrn promoter molecule, and virE operon ribosomalbinding site (RBS) that confers or modulates gene expression from thepromoter. Chimeric promoter without the enhancer molecule was found tobe sufficient for enhanced growth of E. coli cells under selectionpressure. However, the enhancer molecule is required for enhanced growthof Agrobacterium under selection pressure. Other suitable promoterenhancer molecules and RBS molecule could be used in the practice of thepresent invention.

The invention disclosed herein provides polynucleotide moleculescomprising regulatory element fragments that may be used in constructingnovel chimeric regulatory elements. Novel combinations comprisingfragments of these polynucleotide molecules and at least one otherregulatory element or fragment can be constructed and tested in bacteriaand are considered to be within the scope of this invention. Thus, thedesign, construction, and use of chimeric regulatory elements are oneaspect of this invention.

As used herein, the term “fragment,” “fragment thereof,” or “segment”refers in specific embodiments to fragments of a promoter that areprovided, comprising at least about 50, 95, 150, 250, 500, or about 750contiguous nucleotides of a polynucleotide molecule having promoteractivity disclosed herein. These fragments may exhibit promoteractivity, and may be useful alone or in combination with other promotersor promoter fragments, such as in constructing chimeric promoters.

Promoters

Among the gene expression regulatory elements, the promoter plays acentral role. Along the promoter molecule, the transcription machineryis assembled and transcription is initiated. This early step is oftenrate-limiting relative to subsequent stages of protein production.Transcription initiation at the promoter may be regulated in severalways. For example, a promoter may be induced by the presence of aparticular compound or external stimuli, express a gene during aspecific stage of development, or constitutively express a gene. Thus,transcription of a transgene may be regulated by operably linking thecoding sequence to promoters with different regulatory characteristics.Accordingly, a regulatory element such as a promoter, plays a pivotalrole in enhancing the value of a transgenic organism.

As used herein, the term “promoter” refers to a polynucleotide moleculethat is involved in recognition and binding of RNA polymerase and otherproteins such as associated sigma factor and an activator protein toinitiate transcription of an operably linked gene. A promoter may beisolated from the 5′ untranslated region (5′ UTR) of a genomic copy of agene. Alternately, a promoter molecule may be artificially andsynthetically produced or comprise modified DNA sequence. A promoter canbe used as a regulatory element for modulating expression of an operablylinked transcribable polynucleotide molecule. Promoters may themselvescontain sub-elements such as cis-elements or enhancer domains thateffect the transcription of operably linked genes.

In prokaryotes, the promoter consists of two short sequences at −10 and−35 positions upstream from the transcription start site. Sigma factorsnot only help in enhancing RNAP binding to the promoter but helps RNAPtarget which genes to transcribe. The sequence at −10 is called thePribnow box, or the −10 element, and usually consists of the sixnucleotides TATAAT. The Pribnow box is absolutely essential to starttranscription in prokaryotes. The other sequence at −35 (the −35element) usually consists of the six nucleotides TTGACA. Its presenceallows a very high transcription rate. Both of the above consensussequences, while conserved on average, are not found intact in mostpromoters. On average only 3 of the 6 base pairs in each consensussequence is found in any given promoter. It should be noted that theabove promoter sequences are only recognized by the sigma-70 proteinthat interacts with the prokaryotic RNA polymerase. Complexes ofprokaryotic RNA polymerase with other sigma factors recognize totallydifferent core promoter sequences.

Many regulatory elements act in cis (“cis elements”) and are believed toaffect DNA topology, producing local conformations that selectivelyallow or restrict access of RNA polymerase to the DNA template or thatfacilitate selective opening of the double helix at the site oftranscriptional initiation. Cis elements occur within the 5′ UTRassociated with a particular coding sequence, and are often found withinpromoters and promoter modulating sequences (inducible elements). Ciselements can be identified using known cis elements as a target sequenceor target motif using the BLAST programs. Examples of cis-actingelements in the 5′UTR associated with a polynucleotide coding sequenceinclude, but are not limited to, promoters and enhancers.

In prokaryotes, the mRNA translation starts with UTG, GTG or in rarecase UUG, which is usually preceded by sequences characteristic of aribosomal binding site (RBS; Shine and Dalgarno, PNAS 71:1342-1346,1974). The RBS is AG rich and usually is found 6-12 bp before initiationcodon. The RBS is believed to be necessary for efficient mRNAtranslation in bacteria. The Agrobacterium and other Rhizobiaceae 16SP-rrn promoters appear to not contain a consensus RBS to facilitateefficient translation. An additional RBS, for instance from anotheroperon, may be fused to the 3′ end of the P-rrn for efficienttranslation.

In specific embodiments, promoter molecules are provided that exhibit65% or greater identity, 70% or greater identity, 75% or greateridentity, 80% or greater identity, 85% or greater identity, and morepreferably at least 86 or greater, 87 or greater, 88 or greater, 89 orgreater, 90 or greater, 91 or greater, 92 or greater, 93 or greater, 94or greater, 95 or greater, 96 or greater, 97 or greater, 98 or greater,or 99% or greater identity to a polynucleic acid segment selected fromthe group consisting of SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

At least two types of information are useful in predicting promoterregions within a genomic DNA sequence. First, promoters may beidentified on the basis of their sequence “content,” such astranscription factor binding sites and various known promoter motifs.(Stormo, Genome Research 10: 394-397, 2000). Such signals may beidentified by computer programs that identify sites associated withpromoters, such as TATA boxes and transcription factor (TF) bindingsites. Second, promoters may be identified on the basis of their“location,” i.e. their proximity to a known or suspected coding sequence(Stormo). Prokaryotic promoters are typically found within a region ofDNA extending approximately 1-500 basepairs in the 5′ direction from thetranscriptional or translational start codon of a coding sequence. Thus,promoter regions may be identified by locating the translational startcodon of a coding sequence or the transcriptional start site, and movingbeyond the start codon in the 5′ direction to locate the promoterregion.

Promoter sequence may be analyzed for the presence of common promotersequence characteristics, such as a TATA-box and other knowntranscription factor binding site motifs. These motifs are not alwaysfound in every known promoter, nor are they necessary for every promoterto function, but when present, do indicate that a segment of DNA is apromoter sequence.

The activity or strength of a promoter may be measured in terms of theamount of mRNA tRNA, dsRNA, miRNA, rRNA, or protein is specificallyaccumulated during a particular period of time in the growth of a cellcontaining the transgene. An enhanced level of mRNA production may berequired to produce a protein of commercial importance, or a protein forcatalyzing a reaction to produce a compound of commercial importance.Coding sequences for the commercially important proteins can beidentified by those skilled in the art and can be used with thepromoters of the invention to effect production of mRNA and proteinlevels. The activity or strength of a promoter can also be measured interms of enhanced cell growth when a cell is grown under selectionpressure after it has been transformed with, for instance, a gene thatallows for detoxification of a compound under the control of thepromoter.

Regulatory Element Isolation and Modification

Any number of methods well known to those skilled in the art can be usedto isolate a polynucleotide molecule, or fragment thereof, disclosed inthe present invention. For example, is PCR (polymerase chain reaction)technology can be used to amplify flanking regions from a particularstarting nucleotide sequence. A number of methods are known to those ofskill in the art to amplify unknown polynucleotide molecules adjacent toa core region of known polynucleotide sequence. Methods include but arenot limited to inverse PCR (IPCR), vectorette PCR, Y-shaped PCR, andgenome walking approaches. Polynucleotide fragments can also be obtainedby other techniques such as by directly synthesizing the fragment bychemical means, as is commonly practiced by using an automatedoligonucleotide synthesizer. For the present invention, thepolynucleotide molecules were isolated from Agrobacterium genomic DNA bydesigning oligonucleotide primers based on available sequenceinformation and using PCR techniques to extract a particular segment ofDNA.

As used herein, the term “isolated polynucleotide molecule” refers to apolynucleotide molecule at least partially separated from othermolecules normally associated with it in its native state. In oneembodiment, the term “isolated” is also used herein in reference to apolynucleotide molecule that is at least partially separated fromnucleic acids which normally flank the polynucleotide in its nativestate. Thus, polynucleotides fused to regulatory or coding sequenceswith which they are not normally associated, for example as the resultof recombinant techniques, are considered isolated herein. Suchmolecules are considered isolated even when present, for example in thechromosome of a host cell, or in a nucleic acid solution. The term“isolated” as used herein is intended to encompass molecules not presentin their native state.

Those of skill in the art are familiar with the standard resourcematerials that describe specific conditions and procedures for theconstruction, manipulation, and isolation of macromolecules (e.g.,polynucleotide molecules, plasmids, etc.), as well as the generation ofrecombinant organisms and the screening and isolation of polynucleotidemolecules.

Short nucleic acid sequences having the ability to specificallyhybridize to complementary nucleic acid sequences may be produced andutilized in the present invention. These short nucleic acid moleculesmay be used as probes to identify the presence of a complementarynucleic acid sequence in a given sample. Thus, by constructing a nucleicacid probe which is complementary to a small portion of a particularnucleic acid sequence, the presence of that nucleic acid sequence may bedetected and assessed. Use of these probes may greatly facilitate theidentification of transgenic organisms which contain the presentlydisclosed nucleic acid molecules. The probes may also be used to screengenomic libraries for additional nucleic acid sequences related orsharing homology to the presently disclosed promoters. The short nucleicacid sequences may be used as probes and specifically as PCR probes. APCR probe is a nucleic acid molecule capable of initiating a polymeraseactivity while in a double-stranded structure with another nucleic acid.Various methods for determining the structure of PCR probes and PCRtechniques exist in the art. Computer generated searches using programssuch as Primer3, STSPipeline, or GeneUp (Pesole, et al., BioTechniques25:112-123, 1998), for example, can be used to identify potential PCRprimers.

Alternatively, the short nucleic acid sequences may be used asoligonucleotide primers to amplify or mutate a complementary nucleicacid sequence using PCR technology. These primers may also facilitatethe amplification of related complementary nucleic acid sequences (e.g.related nucleic acid sequences from other species).

The primer or probe is generally complementary to a portion of a nucleicacid sequence that is to be identified, amplified, or mutated. Theprimer or probe should be of sufficient length to form a stable andsequence-specific duplex molecule with its complement. The primer orprobe preferably is about 10 to about 200 nucleotides long, morepreferably is about 10 to about 100 nucleotides long, even morepreferably is about 10 to about 50 nucleotides long, and most preferablyis about 14 to about 30 nucleotides long. The primer or probe may beprepared by direct chemical synthesis, by PCR (for example, U.S. Pat.Nos. 4,683,195, and 4,683,202), or by excising the nucleic acid specificfragment from a larger nucleic acid molecule.

Regulatory Elements in a DNA Construct

Various regulatory sequences may be included in a recombinant DNAconstruct. Any such regulatory sequences may be provided in arecombinant vector with other regulatory sequences. Such combinationscan be designed or modified to produce desirable regulatory features.Constructs of the present invention would typically comprise one or moregene expression regulatory elements operably linked to a transcribablepolynucleotide molecule operably linked to an optional 3′ transcriptiontermination polynucleotide molecule. As used herein, the term“heterologous ribosomal binding site” refers to a heterologousnucleotide sequence on mRNA that is bound by the ribosome wheninitiating protein translation.

As used herein, the term “leader” refers to a polynucleotide moleculeisolated from the untranslated 5′ region (5′ UTR) of a genomic copy of agene and defined generally as a segment between the transcription startsite (TSS) and the coding sequence start site. Alternately, leaders maybe synthetically produced or manipulated DNA elements.

DNA Constructs

The DNA constructs of the present invention are generally double Tiplasmid border DNA constructs that have the right border (RB orAGRtu.RB) and left border (LB or AGRtu.LB) regions of the Ti plasmidisolated from Agrobacterium comprising a T-DNA, that along with transfermolecules provided by the Agrobacterium cells, permit the integration ofthe T-DNA into the genome of a plant cell (see for example U.S. Pat. No.6,603,061, herein incorporated by reference in its entirety). Theconstructs may also contain the plasmid backbone DNA segments thatprovide replication function and antibiotic selection in bacterialcells, for example, an Escherichia coli origin of replication such asori322, or ColE1, an Agrobacterium origin of replication, such as oriVor oriRi, and a coding region for a selectable marker such as aadA thatencodes for Tn7 aminoglycoside adenyltransferase (aadA) conferringresistance to spectinomycin or streptomycin, or a gentamicin (Gm, Gent)selectable marker gene. For bacterially mediated plant transformation,the host bacterial strain is often Agrobacterium tumefaciens ABI, C58,EHA101, EHA105, AGLO, or AGLI, or LBA4404, however, other strains knownto those skilled in the art of plant transformation can function in thepresent invention.

As used herein, the term “construct” means any recombinantpolynucleotide molecule such as a plasmid, cosmid, virus, autonomouslyreplicating polynucleotide molecule, phage, or linear or circularsingle-stranded or double-stranded DNA or RNA polynucleotide molecule,derived from any source, capable of genomic integration or autonomousreplication, comprising a polynucleotide molecule where one or morepolynucleotide molecule has been linked in a functionally operativemanner, i.e. operably linked. As used herein, the term “vector” meansany recombinant polynucleotide construct that may be used for thepurpose of transformation, i.e. the introduction of heterologous DNAinto a host cell.

Methods are known in the art for assembling and introducing constructsinto a cell in such a manner that the transcribable polynucleotidemolecule is transcribed into a functional mRNA molecule that istranslated and expressed as a protein product. For the practice of thepresent invention, conventional compositions and methods for preparingand using constructs and host cells are well known to one skilled in theart, see for example, Molecular Cloning: A Laboratory Manual, 3rdedition Volumes 1, 2, and 3 (2000) J. F. Sambrook, D. W. Russell, and N.Irwin, Cold Spring Harbor Laboratory Press). Methods for makingrecombinant vectors particularly suited to plant transformation include,without limitation, those described in U.S. Pat. Nos. 4,971,908,4,940,835, 4,769,061 and 4,757,011, all of which are herein incorporatedby reference in their entirety. These types of vectors have also beenreviewed (Rodriguez, et al. Vectors: A Survey of Molecular CloningVectors and Their Uses, Butterworths, Boston, 1988; Glick et al.,Methods in Plant Molecular Biology and Biotechnology, CRC Press, BocaRaton, Fla., 1993). Typical vectors useful for expression of nucleicacids in higher plants are well known in the art and include vectorsderived from the tumor-inducing (Ti) plasmid of Agrobacteriumtumefaciens (Rogers, et al., Meth. In Enzymol, 153: 253-277, 1987).Other recombinant vectors useful for plant transformation, including thepCaMVCN transfer control vector, have also been described (Fromm et al.,Proc. Natl. Acad. Sci. USA, 82: 5824-5828, 1985).

Transcribable Polynucleotide Molecules

A promoter or chimeric promoter molecule of the present invention may beoperably linked to a transcribable polynucleotide sequence that isheterologous with respect to the promoter molecule. The term“heterologous” refers to the relationship between two or morepolynucleic acid or protein sequences that are derived from differentsources. For example, a promoter or ribosome binding site isheterologous with respect to a transcribable polynucleotide sequence ifsuch a combination is not normally found in nature. In addition, aparticular sequence may be “heterologous” with respect to a cell ororganism into which it is inserted (i.e. does not naturally occur inthat particular cell or organism).

The transcribable polynucleotide molecule may generally be anypolynucleic acid sequence for which an increased level or differentialcell expression of a transcript is desired. Alternatively, theregulatory element and transcribable polynucleotide sequence may bedesigned to down-regulate a specific polynucleic acid sequence. This istypically accomplished by linking the promoter to a transcribablepolynucleotide molecule that is oriented in the antisense direction. Oneof ordinary skill in the art is familiar with such antisense technology.Briefly, as the antisense polynucleic acid molecule is transcribed, ithybridizes to and sequesters a complimentary polynucleic acid moleculeinside the cell. This duplex RNA molecule cannot be translated into aprotein by the cell's translational machinery and is degraded in thecell. Any polynucleic acid molecule may be negatively regulated in thismanner.

A regulatory element of the present invention may also be operablylinked to a modified transcribable polynucleotide molecule that isheterologous with respect to the promoter. The transcribablepolynucleotide molecule may be modified to provide various desirablefeatures. For example, a transcribable polynucleotide molecule may bemodified to increase the content of essential amino acids, to enhancetranslation of the amino acid sequence, to enhance transport of atranslated product to a compartment inside or outside of a cell, or toimprove protein stability, among other effects.

Due to the degeneracy of the genetic code, different nucleotide codonsmay be used to code for a particular amino acid. A host cell oftendisplays a preferred pattern of codon usage. Transcribablepolynucleotide molecules are preferably constructed to utilize the codonusage pattern of the particular host cell. This generally enhances theexpression of the transcribable polynucleotide molecule in a transformedhost cell. Any of the polynucleic acids may be modified to reflect thepreferred codon usage of a host cell or organism in which they arecontained. Modification of a transcribable polynucleotide molecule foroptimal codon usage is described, for example, in U.S. Pat. No.5,689,052.

Additional variations in the transcribable polynucleotide molecules mayencode proteins having equivalent or superior characteristics whencompared to the proteins from which they are engineered. Mutations mayinclude, but are not limited to, deletions, insertions, truncations,substitutions, fusions, shuffling of motif sequences, and the like.Mutations to a transcribable polynucleotide molecule may be introducedin either a specific or random manner, both of which are well known tothose of skill in the art of molecular biology.

Thus, one embodiment of the invention is a chimeric promoter molecule ofthe present invention, such as provided in SEQ ID NOs: 6-8 or variantthereof, operably linked to a transcribable polynucleotide molecule soas to modulate transcription of the transcribable polynucleotidemolecule at a desired level or in developmental pattern uponintroduction of the construct into a cell. In one embodiment, thetranscribable polynucleotide molecule comprises a protein-coding regionof a gene, and the chimeric promoter molecule affects the transcriptionof a functional mRNA molecule that is translated and expressed as aprotein product. In another embodiment, the transcribable polynucleotidemolecule comprises an antisense region of a gene, and the regulatoryelement affects the transcription of an antisense RNA molecule or othersimilar inhibitory RNA in order to inhibit expression of a specific RNAmolecule of interest in a target host cell.

Genes of Commercial Interest

As used herein, the term “gene of commercial interest” refers to atranscribable polynucleotide molecule that includes but is not limitedto a DNA coding sequence or other element intended for expression in thecell, and when expressed in a particular cell or cell type provides adesirable characteristic associated with morphology, physiology, growthand development, exhibiting an effect upon the yield of the cell or cellproduct, nutritional enhancement, or environmental or chemicaltolerance. Suitable transcribable polynucleotide molecules include butare not limited to those encoding traits for desirable biosynthetic,chemical, insecticidal, industrial, nutritional, or pharmaceuticalproperties. For examples, polynucleotide for producing rBGH, antibodies,enzymes for biotechnology applications, and enzymes such as cellulose,glucanase and other hydrolysis enzymes for feed industry. Suitabletranscribable polynucleotide molecules include but are not limited tothose encoding any other agent such as a dsRNA molecule targeting aparticular gene for suppression either within the cell in order to causean effect upon the plant physiology or metabolism or to be provided as apesticidal agent in the diet of a pest that feeds on the cell.

In one embodiment of the invention, a polynucleotide molecule as shownin SEQ ID NOs: 6-8 or variants thereof, is incorporated into a constructsuch that the polynucleotide molecule of the present invention isoperably linked to a transcribable polynucleotide molecule that is agene of commercial interest.

The expression of a gene of commercial interest is desirable in order toconfer a commercially important trait. A gene of commercial interest (atranscribable polynucleotide molecule) that provides a beneficialcommercial trait to a prokaryotic cell may include, for example, but arenot limited to, genes that lead to production of pigments (e.g., melaninby tyrosinase), antibiotics, biofuels (e.g., butanol and pentanol),biodegradable plastics (e.g., PHA; polyhydroxyalkanoate), textiles(e.g., spider silk), to break down of atmospheric pollutants, productionof glucaric acid (e.g., used in synthesis of nylons, and watertreatment), tyrosine (a building block for drugs and food additives),biopolymers, hyaluronic acid (a natural joint lubricant that can be usedto treat arthritis), or isoprenoids (for the biosynthesis of manyimportant pharmaceutical compounds). Other genetic elements may also beutilized, for instance selected from among ones conferring starchproduction (U.S. Pat. Nos. 6,538,181; 6,538,179; 6,538,178; 5,750,876;6,476,295), modified oil production (U.S. Pat. Nos. 6,444,876;6,426,447; 6,380,462), high oil production (U.S. Pat. Nos. 6,495,739;5,608,149; 6,483,008; 6,476,295), modified fatty acid content (U.S. Pat.Nos. 6,828,475; 6,822,141; 6,770,465; 6,706,950; 6,660,849; 6,596,538;6,589,767; 6,537,750; 6,489,461; 6,459,018), biopolymers (U.S. Pat. Nos.RE37,543; 6,228,623; 5,958,745 and U.S. Patent Publication No.US20030028917), pharmaceutical peptides and secretable peptides (U.S.Pat. Nos. 6,812,379; 6,774,283; 6,140,075; 6,080,560), improvedprocessing traits (U.S. Pat. No. 6,476,295), industrial enzymeproduction (U.S. Pat. No. 5,543,576), and biofuel production (U.S. Pat.No. 5,998,700).

Alternatively, a transcribable polynucleotide molecule can effect theabove mentioned cell characteristic or phenotype by encoding a RNAmolecule that causes the targeted inhibition of expression of anendogenous gene, for example via antisense, inhibitory RNA (RNAi), orcosuppression-mediated mechanisms. The RNA could also be a catalytic RNAmolecule (i.e., a ribozyme) engineered to cleave a desired endogenousmRNA product. Thus, any transcribable polynucleotide molecule thatencodes a transcribed RNA molecule that affects a commercially importantphenotype or morphology change of interest may be useful for thepractice of the present invention.

Methods are known in the art for constructing and introducing constructsinto a cell in such a manner that the transcribable polynucleotidemolecule is transcribed into a molecule that is capable of causing genesuppression. For example, posttranscriptional gene suppression using aconstruct with an anti-sense oriented transcribable polynucleotidemolecule to regulate gene expression in cells is disclosed in U.S. Pat.No. 5,107,065 and U.S. Pat. No. 5,759,829; posttranscriptional genesuppression using a construct with a sense-oriented transcribablepolynucleotide molecule to regulate gene expression is disclosed in U.S.Pat. No. 5,283,184 and U.S. Pat. No. 5,231,020. Expression of atranscribable polynucleotide in a cell can also be used to suppresspests feeding on the cell, for example, compositions isolated fromcoleopteran pests (U.S. Patent Application Publication 20070124836,herein incorporated by reference in its entirety) and compositionsisolated from nematode pests (U.S. Patent Application Publication20070250947, herein incorporated by reference in its entirety).

Exemplary transcribable polynucleotide molecules for incorporation intoconstructs of the present invention include, for example, polynucleotidemolecules or genes from a species other than the target species or genesthat originate with or are present in the same species, but areincorporated into recipient cells by genetic engineering methods. Thetype of polynucleotide molecule can include but is not limited to apolynucleotide molecule that is already present in the cell, apolynucleotide molecule from another cell, a polynucleotide moleculefrom a different organism, or a polynucleotide molecule generatedexternally, such as a polynucleotide molecule containing an antisensemessage of a gene, or a polynucleotide molecule encoding an artificial,synthetic, or otherwise modified version of a transgene.

Selectable Markers

As used herein the term “marker” refers to any transcribablepolynucleotide molecule whose expression, or lack thereof, can bescreened for or scored in some way. Marker genes for use in the practiceof the present invention include, but are not limited to transcribablepolynucleotide molecules encoding β-glucuronidase (GUS described in U.S.Pat. No. 5,599,670, which is incorporated herein by reference), greenfluorescent protein and variants thereof (GFP described in U.S. Pat. No.5,491,084 and U.S. Pat. No. 6,146,826, RFP and the like, all of whichare incorporated herein by reference), proteins that confer antibioticresistance, or proteins that confer herbicide tolerance.

Useful antibiotic resistance markers, including those encoding proteinsconferring resistance to kanamycin (nptII), hygromycin B (aph IV),streptomycin or spectinomycin (aad, spec/strep), and gentamycin (aac3and aacC4) are known in the art, among others.

Included within the term “selectable markers” are also genes whichencode a secretable marker whose secretion can be detected as a means ofidentifying or selecting for transformed cells. Examples include markersthat encode a secretable antigen that can be identified by antibodyinteraction, or even secretable enzymes which can be detectedcatalytically. Selectable secreted marker proteins fall into a number ofclasses, including small, diffusible proteins which are detectable,(e.g., by ELISA), small active enzymes which are detectable inextracellular solution (e.g., α-amylase, β-lactamase, phosphinothricintransferase), or proteins which are inserted or trapped in the cell wall(such as proteins which include a leader sequence such as that found inthe expression unit of extension or tobacco PR-S). Other possibleselectable marker genes will be apparent to those of skill in the art.

In specific embodiments, a selectable marker use may be GUS, greenfluorescent protein (GFP), neomycin phosphotransferase II (nptII),luciferase (LUX), an antibiotic resistance coding sequence (aadA), or anherbicide (e.g., glyphosate, dicamba, glufosinate, or 2,4-D) resistancecoding sequence. In certain embodiments, the selectable marker is akanamycin, streptomycin and/or spectinomycin resistance marker. Examplesof coding sequences providing tolerance to antibiotics and herbicidescan be found, for instance, in US Patent Application Publications20080305952 and 20080280361, which are incorporated herein by reference.

Cell Transformation

The invention is also directed to a method of producing transformedcells which comprise, in a 5′ to 3′ orientation, a chimeric promoteroperably linked to a heterologous transcribable polynucleotide molecule.Other molecules may also be introduced into the cell, including 3′transcriptional terminators, 3′ polyadenylation signals, othertranslated or untranslated sequences, transit or targeting sequences,selectable markers, enhancers, and operators.

The term “transformation” refers to the introduction of nucleic acidinto a recipient host. The term “host” refers to prokaryotic cells,especially bacterial cells. As used herein, the term “transformed”refers to a cell or organism into which has been introduced a foreignpolynucleotide molecule, such as a construct. The introducedpolynucleotide molecule may be integrated into the genomic DNA of therecipient cell or organism such that the introduced polynucleotidemolecule is inherited by subsequent progeny or may stay in cytoplasm asa self replicating unit. A “transgenic” or “transformed” cell ororganism also includes progeny of the cell or organism. The term“transgenic” refers to a cell or other organism containing one or moreheterologous polynucleic acid molecules.

There are many methods for introducing heterologous polynucleic acidmolecules into cells. The method generally comprises the steps ofselecting a suitable host cell, transforming the host cell with arecombinant vector, and obtaining the transformed host cell. Suitablemethods for introducing heterologous polynucleic acid molecules intoprokaryotes include Freeze-thaw (Heat shock), triparental mating, andelectroporation, among others. These methods are known to those skilledin the art of prokaryotic transformation.

Any of the above described methods may be utilized to transform a hostcell with one or more gene regulatory elements of the present inventionand one or more transcribable polynucleotide molecules. The host cellmay be any prokaryotic cell, including both Gram negative and Grampositive bacteria. Suitable bacteria include, without limitation,Escherichia sp., Salmonella sp., Klebsiella, Proteus, Yersinia,Azotobacter sp., Pseudomonas sp., Xanthomonas sp., Agrobacterium sp.,Alcaligenes spp., Bordetella sp., Haemophilus influenzae, Methylophilusmethylotrophus, Rhizobium sp., Thiobacillus sp., Streptomyces sp., andClavibacter sp. Preferably, the host cell is selected from among E.coli, Agrobacterium sp., Rhizobium sp., Sinorhizobium sp., Mesorhizobiumsp., Phyllobacterium sp. Ochrobactrum sp. and Bradyrhizobium sp.

The transformed cells are analyzed for the presence of the genes ofinterest and the expression level and/or profile conferred by theregulatory elements of the present invention. Those of skill in the artare aware of the numerous methods available for the analysis oftransformed cells. For example, methods for cell analysis include, butare not limited to Southern blots or northern blots, PCR-basedapproaches, biochemical analyses, phenotypic screening methods, plateassays, and immunodiagnostic assays. The expression of a transcribablepolynucleotide molecule can be measured using TaqMan® (AppliedBiosystems, Foster City, Calif.) reagents and methods as described bythe manufacturer and PCR cycle times determined using the TaqMan®Testing Matrix. Alternatively, the Invader® (Third Wave Technologies,Madison, Wis.) reagents and methods as described by the manufacturer canbe used for transgene expression. Primer sets (pairs of DNA moleculesthat specifically hybridize to a target polynucleotide molecule) aredeveloped to identify specific transcribed transgene sequences. Forexample, the expression of transcript of the present invention can beidentified and measured in the sample using DNA primer molecules or ODat particular absorbance. Primer molecules can be selected by thoseskilled in the art from DNA sequences disclosed herein or other DNAsequences comprising any transgene transcript from which the expressionis driving by a chimeric promoter molecule of the present invention thatmeasures the presence or expression levels of the transgene transcripts.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified. Each periodical, patent, andother document or reference cited herein is herein incorporated byreference in its entirety.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments that are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention, therefore all matter set forth or shown in the accompanyingdrawings is to be interpreted as illustrative and not in a limitingsense.

EXAMPLES Example 1 Identification and Isolation of a 16S rDNA Promoterand Construction of Chimeric P-rrn Promoter Sequences

Four copies of the 16S rDNA are present in A. tumefaciens strain C58.Putative promoter length was determined by aligning four copies of thegene retrieved from Genbank (see FIG. 1) and a consensus sequence wasobtained which was PCR-amplified from the genomic DNA using standardmethods. As the native 16S rDNA promoter does not contain a RibosomalBinding Site (RBS), the promoter was first cloned with a virE operon tooperably link the RBS of the virE operon with the 3′ end of thepromoter.

Xd775: (SEQ ID NO: 9) 5′ GGactagtGGTCTGTTTTTTGACAATTGAATATGAGAAG 3′(P-rrn 5′ forward) Xd776: (SEQ ID NO: 10)5′ cgcatttagcttgatgatcaccatGGCTTTGTTTCTCCTTCAATCATTGACTATTGTCACGTTATTCTG 3′ Xd777: (SEQ ID NO: 11)5′ CgggcccACACTTGAATCGGTAATTTCATTCTAAAGTG 3′ (virE3 reverse)

Primers Xd775 and Xd776 were used to amplify P-rrn from the C58 genomicDNA using the pfu polymerase. After 10 PCR cycles, the amplified DNA wasmixed with pTiC58 DNA comprising virE operon and Xd777 and amplified foradditional 5 cycles with 4.5 min elongation time. One μl of the productwas further amplified with the primers Xd775 and Xd777 to amplify DNAcomprising the P-rrn and virE1 to virE3 (4.3 kb) coding region. Theamplified fragment was gel isolated and cloned into pCR® Blunt II TOPO®cloning vector (Invitrogen), which resulted in the intermediate plasmidXXYE02.0337. The cloned DNA was confirmed by sequencing.

To replace the virE promoter in ABI strain in vivo, pMON96945 wasconstructed. pMON83948, which contained virE flanking sequence, wasPCR-amplified with the primers Xd778 and Xd779 using pfu polymerase. ThePCR product was digested with ApaI/SpeI and ligated with the Xd775 andXd777 amplified PCR fragment from the intermediate vector XXYE02.0337digested with the same enzymes. The resultant plasmid pMON96945 (FIG. 5)was confirmed by sequencing.

Xd778: (SEQ ID NO: 12) 5′ GGactagtCAGAAATTACGATTTTCCTAGTGCCTTC 3′ Xd779:(SEQ ID NO: 13) 5′ ATGCCAATAGgggcccAATATCGGCATTTTCTTTTGCGTTTTTATTT G 3′

To replace the native virE operon promoter in A. tumefaciens ABI strain(a C58 derivative) with the identified strong constitutive P-rrnpromoter, pMON96945 was electroporated into the Agrobacterium competentcells and selected on carbenicillin 50 mg/l for integration of P-rrnpromoter by homologous recombination. The carbenicillin resistantcolonies was shaken at 28° C. for 2-3 hours and plated onto LB mediumwith 5% sucrose for the 2nd crossover. The carbenicillin sensitivecolonies were checked for the replacement of the P-rrn-virE by PCR usingthe following junction primers:

(SEQ ID NO: 14) 5′ GGTCTGTTTTTTGACAATTGAATATGAGAAG 3′ (P-rrn 5′) Xd837(SEQ ID NO: 15) 5′ GAGTCGGGCTTCCGTGCATGTTG 3′ (virE2 mid reverse) Xd838(SEQ ID NO: 16) 5′ AATGCACGGTGATGATGTTGATCG 3′ (virE upstream flanking)Xd839 (SEQ ID NO: 17) 5′ TGATCACCATggCtTTGTTTCTCC 3′ (P-rrn-virE13′reverse) Xd840

The primers Xd839 and Xd838 amplified a 970 bp fragment from themodified ABI strain and primers Xd839 and Xd838 amplified a 759 bpfragment from the unmodified ABI strain. The modified strain wasdesignated as AB8. The P-rrn-virE operon was constructed using pMON96945knockout plasmid. The replacement of the virE operon promoter wasconfirmed by sequencing the PCR product. Other C58 derived strains withdifferent improvements can be made using the same strategy. Overexpression of the virE operon has been found is known to be useful intransferring large DNA segments to a host cell.

Example 2 Construction of P-rrn-aadA Prokaryotic Expression Cassette forImproving Growth of Bacteria Carrying Low Copy Plasmids in Agrobacterium

The oriRi vectors (US20070074314) grow slower than oriV vectors underantibiotic selection due to the gene dose effect of the low or multiplecopy vectors in Agrobacterium. To construct a high expressing aadAcassette, the P-rrn with virE RBS was re-amplified with the primersXd1021 and Xd776 using pfu polymerase and pMON96945 as a template.

Xd1021: (SEQ ID NO: 18) 5′ GGactagtcatgaGGTCTGTTTTTTGACAATTGAATATGAGAAG3′ Xd776: (SEQ ID NO: 19)5′ CGCATTTAGCTTGATGATCACCATggCtTTGTTTCTCCTTCAATCATTGACTATTGTCACGTTATTCTG 3′

The amplified PCR product was digested with BspHI and NcoI (underlinedin primers), gel purified, and ligated into pMON83934 digested withBspHI. The ligation mixture was electroporated into A. tumefaciens ABIcompetent cells, plated onto LB medium with 100 mg/l spectinomycin andcultured at 30° C. After 2 days large colonies was transferred into LBmedium containing 50 mg/l spectinomycin and shaken at 30° C. overnight.The resultant plasmid pMON107330 was rescued into E. coli cells,mini-prepared, and confirmed by sequencing.

A 2T-DNA vector pMON107336 was made by replacing the native bacterialspectinomycin selection cassette of 2T vector pMON107333 (opened withSpeI blunted/BspHI) with the P-rrn-aadA cassette from pMON107330 excisedwith PstI blunted/BspHI.

Both pMON107330 (1T oriRi) and pMON107336 (2T oriRi) contained the longversion of P-rrn promoter shown in FIG. 2. Due to a HindIII site presentin the P-rrn promoter, which is frequently used as restriction site forsubcloning, the long version of P-rrn from pMON107330 was re-amplifiedwith Xd1033 and Xd1034 to remove HindIII using the primers Xd1033 andXd1034. The PCR fragment was digested with BspHI/NcoI, gel purified, andinserted into pMON83934 (US20070074314) opened with BspHI, whichresulted in the 1 T-DNA vector pMON107331. The short version P-rrn inpMON107331 (1T oriRi) is shown in FIG. 3.

Xd1033: (SEQ ID NO: 20) 5′ GATTTTGGtcatgaGCGGGACCTGGAGAGATTTGGGTCCTAGTG3′ Xd1034: (SEQ ID NO: 9) 5′ aggcatgcaagcttGATGGGGATCAGATTGTCGTTTCC 3′

Example 3 Testing the Chimeric P-rrn Promoter Function in E. coli

Plasmids carrying long or short versions of P-rrn and the aadA gene wereintroduced into E. coli TOP10 by electroporation and selected on LBmedium containing 50 mg/l spectinomycin. The E. coli colonies werepicked and grown in LB containing 100 mg/l spectinomycin. As seen in theFIG. 10, growth rates for E. coli carrying either longer or shorterversion of P-rrn with aadA were higher than the construct carrying theaadA gene with its own promoter. It took less than 5 hours at thespectinomycin concentration of 50 mg/l or 6 hours at spectinomycinconcentration of 100 mg/l to grow E. coli cells to a desirable level.The time savings indicated that the cloning process could be improvedusing these sequences. The growth rate of E. coli with the P-rrn-aadAcassettes was significantly faster that the native aadA cassette at 100mg/l spectinomycin, indicating that the P-rrn-aadA is more highlyexpressed than the native cassette, and provides higher protection fromthe antibiotic selection.

Example 4 Testing the Chimeric P-rrn Promoter Function in Agrobacterium

Plasmids carrying the long or the short versions of P-rrn and the aadAgene were introduced into Agrobacterium ABI by electroporation andselected on LB medium containing 50 mg/l spectinomycin. Three colonieswere inoculated into 3 ml LB with various spectinomycin concentrationsas shown in FIG. 11, and cultures were shaken at 250 rpm at 28° for 17hours after which absorbance was measured at OD600.

As shown in FIG. 11, when the constructs carrying chimeric P-rrn-aadAcassettes were transferred into Agrobacterium and Agrobacterium wasgrown, it was surprisingly found that Agrobacterium carrying thepMON107331 construct containing the short P-rrn promoter with aadAshowed significantly reduced growth. This may indicate that requirementsfor promoter strength are different in E. coli and Agrobacterium. Thepresence of a cis enhancer element or additional Agrobacterium-specificregulatory sequences located in the 51 bp upstream of the HindIII in theP-rrn was also found.

The oriRi vector with the long version of P-rrn resist a wide range ofspectinomycin and grows fairly well at 300 mg/l spectinomycin, while thewild type aadA cassette in an oriRi vector (See U.S. Patent ApplicationPublication 2007/0074314, incorporated herein by reference) showedsignificant growth reduction at spec 75 mg/l. Although the oriRi vectorsreplicated in Agrobacterium as 1-2 copies per chromosome and the oriVvector replicates at approximately 10 copies per chromosome, theAgrobacterium growth rate with oriRi vectors was 0.1-0.2 OD better thanthe oriV vector in all spectinomycin concentrations tested. Since thespectinomycin resistance level showed a linear relationship with plasmidcopy number (U.S. Patent Application Publication 2007/0074314), it wasestimated that the P-rrn promoter strength is at least 10 times strongerthan the native aadA promoter.

Example 5 Deletion of HindIII Site from the P-rrn Promoter Retains theFull P-rrn Activity

Since 51 bp upstream of HindIII site were responsible for highexpression of P-rrn in Agrobacterium and because the HindIII site isuseful for subcloning, the HindIII site was filled-in with T4 DNApolymerase in the long version of P-rrn to remove it (FIG. 4) as well asto retain the P-rrn promoter strength in Agrobacterium. For thispurpose, the 2T-DNA vector pMON107336 was used since it contains only asingle HindIII site. After filling-in, the vector was self-ligated andtransformed into E. coli TOP10 cells (Invtrogen). Colonies fromovernight LB plate with 50 mg/l spectinomycin were picked and culturedin 2 ml LB with 50 mg/ml spectinomycin for 5 hours. The mini-preparedplasmid was digested with HindIII/PstI to confirm the loss of HindIIIsite. The resulting plasmid pMON107337 was confirmed by sequencing andtransferred into Agrobacterium to test the spectinomycin resistancelevel as described above. It was confirmed that the HindIII minus P-rrnpromoter has the same level of spectinomycin resistance as shown in FIG.12.

The 51 bp HindIII upstream fragment is conserved in A. tumefaciens asfound by the alignment of the consensus P-rrn sequence from C58 andMAFF301001 strains. This indicates that the 51 bp fragment is useful forenhanced function in Agrobacterium cells.

Example 6 Identification of P-rrn Sequences from Other Rhizobiales

P-rrn promoters from a number of bacteria belonging to the Rhizobialesand Rhizobiaceae were identified by aligning operons/genes for 16s rRNAsavailable in the public databases. The accession numbers for these areas follows: Agrobacterium tumefaciens C58 (Genbank accessions AE009201,AE008688; AE008980, AE008688; AE009324, AE008689; AE009348, AE008689),Rhizobium leguminosarum bv. viciae 3841 (complete genome; Genbankaccession NC_(—)008380), Rhizobium etli CFN 42 (complete genome; Genbankaccession NC_(—)007761), Sinorhizobium medicae WSM419 (Genbank accessionCP000738), and Sinorhizobium meliloti 1021 (Genbank accessionNC_(—)003047). The identified P-rrn promoters from each bacteria and theconsensus sequences are provided as SEQ ID NOs: 1-5 and 22-37 (seedescription of sequence listing). Alignment of consensus sequences ofall P-rrn promoters from these organisms showed about 70% identity. EachP-rrn promoter is operably linked to a RBS sequence which is operablylinked to a transcribable sequence to produce an expressionunit/cassette which is then inserted in a vector as described elsewherein the specification. The vector is then transformed into a host cell toenhance the expression of the transcribable sequence in the host cell.

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims. All publications and publishedpatent documents cited in this specification are incorporated herein byreference to the same extent as if each individual publication or patentapplication is specifically and individually indicated to beincorporated by reference.

1. A polynucleotide molecule comprising a 16S rDNA promoter moleculeoperably linked to a nucleic acid comprising a heterologous ribosomalbinding site, wherein the polynucleotide molecule has promoter activity.2. The polynucleotide molecule of claim 1, wherein the 16S rDNA promotermolecule is isolated from a prokaryote.
 3. The polynucleotide moleculeof claim 1, wherein the 16S rDNA promoter molecule comprises a nucleicacid sequence selected from the group consisting of: a) a nucleic acidsequence comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ IDNO:4, or any of SEQ ID NOs:22-37; b) a nucleic acid sequence comprisingat least 65% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,or SEQ ID NO:4, or any of SEQ ID NOs:22-37, wherein the nucleic acidsequence comprises promoter activity; and c) a fragment of the nucleicacid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4,or any of SEQ ID NOs:22-37, wherein the fragment has promoter activity.4. The polynucleotide molecule of claim 2, wherein the prokaryote is abacterium.
 5. The polynucleotide molecule of claim 4, wherein thebacterium is a member of the Rhizobiales.
 6. The polynucleotide moleculeof claim 5, wherein the member of the Rhizobiales is selected from thegroup consisting of: Rhizobium sp., Sinorhizobium sp., Mesorhizobiumsp., Phyllobacterium sp., Ochrobactrum sp., and Bradyrhizobium sp. 7.The polynucleotide molecule of claim 1, wherein the polynucleotidecomprises a sequence selected from the group consisting of: a) a nucleicacid sequence comprising SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8; b) anucleic acid sequence comprising at least 65% sequence identity to SEQID NO:6, SEQ ID NO:7 or SEQ ID NO:8; and c) a fragment of the nucleicacid sequence of SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.
 8. Thepolynucleotide molecule of claim 7, wherein the polynucleotide comprisesat least about 65%, at least about 85%, at least about 90%, at leastabout 95% identity, or at least about 98% identity to the nucleic acidsequence of SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.
 9. Thepolynucleotide molecule of claim 7, comprising the sequence of SEQ IDNO:6, SEQ ID NO:7 or SEQ ID NO:8
 10. The polynucleotide molecule ofclaim 7, comprising a fragment of the nucleic acid sequence of SEQ IDNO:6, SEQ ID NO:7 or SEQ ID NO:8.
 11. The polynucleotide molecule ofclaim 1, further defined as operably linked to a heterologoustranscribable polynucleotide molecule.
 12. The polynucleotide moleculeof claim 11, wherein the heterologous transcribable polynucleotidemolecule encodes a selectable marker.
 13. The polynucleotide molecule ofclaim 12, wherein the selectable marker confers resistance to aselective agent selected from the group consisting of: kanamycin,spectinomycin, streptomycin, hygromycin, gentamycin, glyphosate,dicamba, and glufosinate.
 14. The polynucleotide molecule of claim 12,wherein the selectable marker is aadA.
 15. The polynucleotide moleculeof claim 1, wherein the ribosomal binding site is isolated from theAgrobacterium virE operon.
 16. A transgenic cell transformed with thepolynucleotide molecule of claim
 1. 17. The transgenic cell of claim 16,wherein the cell is a prokaryotic cell.
 18. The transgenic cell of claim17, wherein the prokaryotic cell is a bacterial cell.
 19. The transgeniccell of claim 18, wherein the bacterial cell is a member of theRhizobiales.
 20. The transgenic cell of claim 19, wherein the member ofthe Rhizobiales is selected from the group consisting of: Rhizobiumspp., Sinorhizobium spp., Mesorhizobium spp., Phyllobacterium spp.,Ochrobactrum spp., and Bradyrhizobium spp.
 21. The transgenic cell ofclaim 18, wherein the bacterial cell is an E. coli cell.
 22. Atransgenic organelle comprising the polynucleotide molecule of claim 1.23. A recombinant Agrobacterium cell wherein the function of the nativevirB operon, virC operon, virD operon, or virE operon promoter of thecell has been replaced with a heterologous constitutive promoter. 24.The Agrobacterium cell of claim 23, wherein the promoter comprises thepolynucleotide molecule of claim
 1. 25. A method for enhancingexpression of a transgene in a prokaryotic cell comprising: (a)transforming the cell with a transgene operably linked to a 16S rDNApromoter operably linked to a ribosomal binding site; (b) growing thecell; and (c) testing the cell for enhanced expression of the transgene.26. The method of claim 25, wherein the transgene confers a commerciallyimportant trait.
 27. The method of claim 25, further comprising the stepof: (d) harvesting a product of the enhanced expression of thetransgene.
 28. The method of claim 27, wherein the product of theenhanced transgene expression is a protein or a compound.